Fuchs Endothelial Corneal Dystrophy (FECD), a common age-related dystrophy, which is more prevalent in women, is of unknown etiology. In FECD, corneal endothelial (CE) cell loss is accompanied by abnormal extracellular matrix (ECM) deposition in the form of guttae. Our laboratory was the first to link oxidative stress with FECD pathogenesis. Specifically, we showed that in FECD, oxidant-antioxidant imbalance due to suboptimal Nrf2-regulated antioxidant defense, including a decline in its transcriptional target NQO1, leads to oxidative DNA damage, mitochondrial dysfunction, and apoptosis. However, little is known regarding the mechanism of guttae formation and the predominance of FECD development in women. The mechanism of the observed DNA damage in cellular degeneration in FECD is not known. Building upon our previous findings, we propose to investigate if oxidant-antioxidant imbalance in FECD leads to endothelial mesenchymal transition (EMT) with concomitant ECM deposition, and if this imbalance causes the formation of cytotoxic estrogen metabolites, which are known to accumulate in tissues with the defective Nrf2-NQO1 and cause DNA damage, more commonly in women. Since our preliminary studies indicate a defective DNA damage response (DDR) in FECD, we will determine whether it leads to decreased DNA damage repair, cellular senescence, and apoptosis. Our proposal seeks to explore three different disease mechanisms: EMT, estrogen genotoxicity, and DDR, all of which either independently or collectively may lead to the development of FECD. Our study is significant, as the investigation of the mechanisms involved in the oxidative stress-induced cellular damage will provide new treatment targets for FECD. In order to achieve these aims, we will use our newly developed telomerase-immortalized human CE cells and the in vivo oxidative stress model based on the ultraviolet-A irradiation of mouse corneas. This model is specifically relevant to our study since it replicates in mice the CE morphological changes with guttae-like lesions and apoptosis, characteristically seen in FECD patients. Our Specific Aims are: Aim 1: Determine whether oxidant-antioxidant imbalance seen in FECD activates EMT that leads to aberrant ECM deposition seen in guttae. This aim is based on the hypothesis that in FECD chronic oxidative stress causes endothelial cells to undergo EMT and enhanced ECM deposition leading to guttae formation that can be modified by activation of Nrf2 pathway. Aim 2: Investigate the role of reactive estrogen metabolites in initiating an estrogen genotoxic pathway that may account for higher incidence and severity of FECD in women. This aim is based on the hypothesis that altered levels of estrogen-metabolizing enzymes lead to imbalanced estrogen metabolism and increased formation of depurinating estrogen-DNA adducts in FECD. Aim 3: Investigate the role of DDR on mitochondrial and nuclear DNA repair during oxidative stress- induced damage of CE in FECD. This aim is based on the hypothesis that impaired DDR leads to deficient DNA repair that results in mitochondrial dysfunction, cellular senescence, and apoptosis.